JP2020529861A - Biosynthesis of eriodictyol from engineered microorganisms - Google Patents

Abstract

The present invention relates to the production of eriodictyol by biological conversion.

Description

Cross-reference to related applications This application claims the priority of US Provisional Patent Application No. 62 / 542,843 entitled "BIOSYNTHESIS OF ERIODICTYOL FROM ENGINEERED MICROBES" filed on August 9, 2017. All of the disclosures are incorporated herein by reference.

The art of the present invention relates to methods and processes useful for the production of eriodictyol from precursor molecules, particularly naringenin. More specifically, the production of eriodictyol from naringenin via enzymatic conversion in vivo.

The present invention focuses on the conversion of naringenin to eriodictyol. The present disclosure relates to the synthesis of eriodictyol by microbial fermentation.

Flavonoids are one of the largest number of structurally diverse natural products in the plant kingdom. They are known to have a variety of beneficial medicinal and chemopreventive effects on human health. Flavonoids have been shown to act as antioxidants, antibacterial agents, anti-inflammatory agents, and their anti-cancer properties have been demonstrated. However, the low water solubility and overall instability of these compounds limits their pharmaceutical use. Chemically, the flavone is 2-phenyl-4H-1-benzopyran-4-one, the hydroxyl group of which may or may not be present at various positions on the ring. An example of a flavone is apigenin, whose chemical name is 2- (p-hydroxyphenyl) -4H-1- (5,7-dihydroxybenzopyran-4-one. Therefore, this definition includes flavans, Flavan-3-ols (catechols), flavans 3,4-diols (leucanthocyanidins), flavones, flavonols, and flavonones are included in the conventional sense. The flavones of interest in the present invention are naringin, naringenin, and eriodicthiol. Is.

Flavonoids are secondary plant metabolites synthesized using the phenylpropanoid pathway (Winkel-Sirley, 2001) and are present in a wide variety of plants. Flavonoid-derived compounds have received a lot of attention due to their use as health-promoting ingredients in the human diet. This is due to a variety of their chemotherapeutic, antioxidant, and anti-asthma activities (Bravo 1998; Lamartiniere 2000; File et al., 2001; and Le Marchand 2002). However, the exact profile and amount of certain flavonoids in plants varies widely from species to species.

As mentioned above, flavonoids are important natural compounds with diverse biological activities. Citrus-derived flavonoids make up a series of potentially medically important flavonoids. Naringenin belongs to this series of flavonoids and has been found to exhibit potent anti-inflammatory and antioxidant activity in mammalian systems. Studies of several strains suggest that flavonoids, naringin, and naringin supplements from other citrus fruits are beneficial in the treatment of obesity, diabetes, hypertension, and metabolic syndrome in humans. Several molecular mechanisms underlying these beneficial activities have been elucidated. However, the mechanism of their effects on obesity and metabolic disorders has not yet been fully elucidated.

Naringin Naringin is a flavanone-7-O-glycoside that can be enzymatically modified to naringenin. Naringenin is naturally present in citrus fruits, especially grapefruit, where naringin is involved in the bitterness of the fruit. In the production of commercial grapefruit juice, the enzyme naringinase can be used to eliminate the bitterness caused by naringin.

Naringin can inhibit several drug-metabolizing cytochrome P450 enzymes, including CYP3A4 and CYP1A2, and cause systemic drug-drug interactions. Ingestion of naringin and related flavonoids also affects the intestinal absorption of certain drugs, which can result in either an increase or decrease in circulating drug levels. Taking citrus fruits (especially grapefruit) and other juices with the drug to avoid interfering with the absorption and metabolism of the drug is contraindicated in patients with drugs that may be affected by citrus flavonoids. ..

Naringenin Naringenin is a flavanone and can be found in grapefruit, orange and tomato skins. This bioflavonoid is difficult to absorb by ingestion. However, it has been shown to have an inhibitory effect on human cytochrome P450 isoform CYP1A2 when ingested. This beneficial property can be extended to the extent that it is a potent inhibitor of the benzo (a) pyrene metabolizing enzyme benzo (a) pyrene hydroxylase (AHH). Similarly, naringenin has been shown to reduce oxidative damage to DNA in in vitro and animal studies. Naringenin has also been shown to reduce the production of hepatitis C virus by infected hepatocytes (liver cells) in cell culture. This appears to be second only to naringenin's ability to inhibit the secretion of very low density lipoproteins by cells. Naringenin also protects LDLR-deficient mice from the effects of obesity on a high-fat diet. Naringenin lowers plasma and hepatic cholesterol levels by suppressing HMG-CoA reductase and ACAT in rats fed a high cholesterol diet.

Eriodictyol Eriodictyol is also a flavonoid compound found in some plants. Compared to naringenin, it has additional hydroxylation. It also has higher biological activity. Eriodictyol can shield bitterness and is widely used in the manufacture of pharmaceuticals and foods.

In the extraction industry, eriodictyol is harvested from the Eriodictyon califormium plant (Ley et al., 2005). When used in trials, eriodictyol has been demonstrated to significantly reduce the bitterness of caffeine without exhibiting a strong flavor or taste characteristic inherent in it, which is the taste of food, beverages, and pharmaceuticals. It had great potential for profile modification. In addition, eriodictyol has been shown to possess higher biological activity and greater anti-cancer activity than many other flavonoids (Lee et al., 2007; Chu et al., 2016). For anti-cancer effects, eriodicthiol was thought to have little or no effect on normal cultured human cells (Matsuo et al., 2005), but apoptotic DNA ladder formation, chromatin in HL-60 leukemia cells. It has been shown to induce condensation and cytotoxic toxicity (Ogata 2000).

As a secondary metabolite, flavonoids are often produced in small or variable amounts in certain plant species, thus hindering cost-effective isolation and widespread use. In addition, some of these species are endangered in their natural habitat, further limiting the availability of some plant metabolites for commercial use.

With respect to its additional usefulness in the pharmaceutical field, eriodictyol has been found to be a TRPV1 antagonist and can be used as an analgesic. The vanilloid 1 receptor (TRPV1) is a calcium-permeable channel involved in the transmission and modulation of acute and chronic pain signaling. As such, this receptor is a potential target for the treatment of some pain disorders.

In plants, it is known that eriodictyol can be induced by hydroxylation of plant naringenin catalyzed by flavonoid 3'-hydroxylase (F3'H), which is a cytochrome P450-dependent monooxygenase (Brugliera et al., 1999; Kaltenbach et al., 1999). In the last few decades, biocatalytic hydroxylation of naringenin has been achieved by the identification and engineering of several cytochrome P450 hydroxylases from plants and microorganisms (Kasai et al., 2009; Amor et al., 2010; Chu et al., 2016). However, because P450 hydroxylase is a membrane-binding protein, its activity depends on P450 reductase and heme biosynthesis, which makes functional expression of P450 in prokaryotic systems difficult (Oeda et al., 1985). .. Recently, efforts have been made to identify non-P450 hydroxylases for the biological conversion of naringenin to eriodictyol. Lin and Yan (2014) have described E. coli. We found that HpaBC, first identified as a two-component monooxygenase that catalyzes orthohydroxylation of 4-hydroxyphenylacetate in Coli, can hydroxylate naringenin to eriodictyol (Lin and Yan 2014). Lee et al. (2014) have shown that SAM5, a monooxygenase from Saccharothlix espanaensis that catalyzes the hydroxylation of caffeic acid to ferulic acid, has activity on naringenin. However, the reported titers of these non-P450 hydroxylase-mediated eriodictyol are low for use in scaled-up production. With the expressed SAM5 enzyme alone, E.I. It showed low activity against flavonoids in E. coli cells. Co-expression of P450 reductase was one way to increase activity. However, the stimulation of flavonoid hydroxylation by this method was limited, with only an enhancement of about 34-50% observed (Lee et al., 2014).

Taken together, the purpose of this disclosure is to demonstrate the production of naringenin and eriodictyol by biotransformation, and the increased amounts of such compounds are available for research in the fields of industrial use and pharmaceutical and functional cosmetics. To provide the technology to be.

Thus, organisms that can carry the required enzymes and modify the natural enzymes or certain microorganisms to use commercially significant fermentation processes to specifically increase the production of the flavonoids of interest. The conversion better addresses the limited quality and supply issues of eriodictyol.

According to the present invention, a practical approach to improving the production of eriodictyol allows for its economical production by biotransformation. In addition, among plants known to produce certain flavonoids, the composition and amount vary widely with weather conditions and geography, so they can be used as medicinal foods or as precursors for the development of new drugs. It is difficult to obtain sufficient amounts of specific flavonoids for use. One of the practical and promising approaches to address this problem and provide sufficient amounts of bioactive flavonoids is in vivo conversion by biotransformation (Lim et al., 2004; Kim et al., 2005). This reverses the limited availability and high cost of procuring these compounds from the plant kingdom. This relatively increased abundance allows the production of several flavonoids for use in the food, pharmaceutical and cosmetics industries.

Therefore, the flavonoids provided herein need to be developed commercially and such compounds are needed to utilize relatively common starting substrates such as naringin as a starting molecule. As such, such production of desirable flavonoids can be as commercially cost-effective as possible. The present disclosure provides a method of producing eriodictyol from naringin and / or naringenin.

In addition, plant extraction processes typically utilize solid-liquid extraction techniques that use solvents such as hexane, chloroform, and ethanol for recovery. However, solvent extraction is itself energy intensive, poses a problem of toxic waste treatment, requires a large acreage for the plant itself to grow, and requires further purification of the trace components recovered. Produce products that do. Therefore, new production methods are also needed to reduce the cost of producing eriodictyol and reduce the environmental impact of large-scale cultivation and processing. One such potential solution is a fermented biotransformation that allows production in a particular microbial species, increasing the selectivity, abundance, and purity of the desired commercially available eriodictyol. The use of technology.

In addition to the above, consumers approve and actively seek natural and biological sources of food, feed, flavor or medicinal ingredients, while consumers are sources of source, consistent taste. We are also concerned about profiles and environmentally sustainable production. In this context, the microbial fermentation and production methods of the present invention provide the flavonoids of the present invention in amounts useful for various industries and studies in a more natural manner than inorganic synthesis or current plant extraction techniques.

Therefore, there is a need to develop new methods for producing eriodictyol economically and conveniently to further enable industrial use and consumption by humans.

The present invention is a modified E.I. Includes methods of producing eriodictyol from naringenin via E. coli or other microbial strains.

In particular, the present invention provides the production of eriodictyol by biological conversion.

The method provides an approach for the biosynthesis of eriodictyol in microbial cultures using specific synthetic pathways.

In terms of product / commercial utility, several products containing eriodictyol are on the market in the United States and can be used for all purposes, from analgesics to pest repellents, as well as food and dietary supplements. Products containing eriodictyol can be aerosol, liquid, or granular formulations.

With respect to the cell line in the embodiment, it allows gene transformation with a gene selected or selected from the group consisting of bacteria, yeast, and combinations thereof, followed by the biosynthetic production of eriodictyol from naringenin. Is any cell line that allows for. In the most preferred microbial system, E.I. E. coli is used to produce eriodictyol and dihydroquercetin ((“DHQ”) or taxifolin).

The present disclosure is susceptible to various modified and alternative forms, but those particular embodiments are shown in the drawings by way of example and are described in detail herein. However, the drawings and detailed description presented herein are not intended to limit this disclosure to the particular embodiments disclosed, the intent of which is defined by the appended claims. To include all modifications, equivalents, and alternatives that fall within the spirit and scope of the present disclosure.

Other features and advantages of the invention will become apparent in the following detailed description of preferred embodiments of the invention with reference to the accompanying drawings.

A plasmid map of Sam5-pRSFDuet with major components is shown. HPLC analysis of the biological conversion of naringenin to eriodictyol using the ERI-01 strain. FIG. 3 LC-MS analysis of the biological conversion of naringenin using the ERI-01 strain. FIG. 3 LC-MS analysis of the biological conversion of naringenin using the ERI-01 strain. Sequence alignment of HpaC, PpFR, and SeFR. SDS-PAGE of purified flavin reductase. Lanes 1, 3 and 5 are protein markers and their corresponding sizes are listed on the left. Purified flavin reductase is indicated by the red arrow. Lane 2: SeFR; Lane 4: PpFR, and Lane 6: HpaC. Relative enzymatic activity of FAD reductase compared to HpaC activity. Maps of plasmids for HpaC-pCDF, PpFR-pCDF, and SeFR-pCDF. Production of eriodictyol in strains ER01-ER05 as measured by HPLC. ER production of each of the ER01-ER05 strains. A plasmid map containing each of the isolated flavin reductase, HpaC, PpFR, and SeFR is shown. Production of eriodictyol using engineered strains of ERI-06, ERI-07, and ERI-08. The molecular structure of naringenin is shown. C ₁₅ H ₁₂ O ₅ ; Average mass: 272.253 Da; Naringenin-IUPAC name 5,7-Dihydroxy-2- (4-hydroxyphenyl) chroman-4-one. Shows the molecular structure for eriodictyol; C ₁₅ H ₁₂ O ₆ ; average mass: 288.252 Da; eriodictyol-IUPAC name: (2S) -2- (3,4-dihydroxyphenyl) -5,7 -Dihydroxy-4-chromanone.

Description of Preferred Embodiments Description of terms used herein:
Definition:
The cell line is any cell that provides expression of ectopic proteins. It included bacteria, yeast, plant cells, and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes in vitro expression of proteins based on cellular components such as ribosomes.

A "coding sequence" gives one of ordinary skill in the art its usual and customary meaning, which is used without limitation to refer to a DNA sequence encoding a particular amino acid sequence.

Cell line growth. Growth involves providing a suitable medium that allows the growth and division of cells. It also includes providing resources that allow cells or cell components to be translated to produce recombinant proteins.

Protein expression. Protein production can occur after gene expression. It is composed at a stage after DNA is transcribed into messenger RNA (mRNA). The mRNA is then translated into a polypeptide chain and finally folded into a protein. DNA is present in cells through transfection-the process of deliberately introducing nucleic acids into cells. This term is often used for non-viral methods in eukaryotic cells. It can also refer to other methods and cell types, but other terms are preferred: "transformation" is used to describe nonviral DNA transfer in non-animal eukaryotic cells, including bacterial, plant cells. Used more often. In animal cells, transfection is the preferred term because transfection is also used to refer to the progression of these cells to a cancerous state (carcinogenesis). Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infections are included in the definition of transfection herein.

yeast. According to the present invention, the yeast claimed herein is a eukaryotic unicellular microorganism classified as a member of the fungal community. Yeast is a unicellular organism that evolved from a multicellular ancestor, but species useful in the present invention have the ability to develop multicellular properties by forming strings of linked budding cells known as pseudohyphae or pseudohyphae. To have.

Structural terms:
As used herein, the singular forms "a", "an", and "the" include references to multiple unless the content clearly indicates otherwise.

As long as terms such as "contain" and "have" are used herein or in the claims, such terms are interpreted as "contains" when used as a transitional term in the claims. It is intended to be inclusive of the term "contains" in a similar manner.

The term "exemplary" is used herein to mean "useful as an example, case, or example." The embodiments described herein as "exemplary" do not necessarily have to be considered preferred or advantageous over other embodiments.

The term "complementary" gives those skilled in the art its usual and customary meaning and is used without limitation to describe the relationships between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, dependent techniques also include isolated nucleic acid fragments complementary to the complete sequences reported in the attached sequence listing and their substantially similar nucleic acid sequences.

The terms "nucleic acid" and "nucleotide" give those skilled in the art their usual and customary meanings of deoxyribonucleotides or ribonucleotides, and their polymers in either single- or double-stranded form. Used without restrictions to point. Unless otherwise specified, the term includes nucleic acids that have binding properties similar to reference nucleic acids and contain known analogs of naturally occurring nucleotides that are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, specific nucleic acid sequences imply not only the sequences explicitly shown, but also their conservatively modified or degenerate variants (eg, degenerate codon substitutions) and complementary sequences. Include.

The term "isolated" gives those skilled in the art its usual and conventional meaning, and when used in the context of isolated nucleic acids or isolated polypeptides, by human hands. , Used without limitation to refer to nucleic acids or polypeptides that exist apart from their natural environment and are therefore not products of nature. The isolated nucleic acid or polypeptide can be present in purified form or in a non-natural environment such as a transgenic host cell.

As used herein, the terms "incubate" and "incubate" are conditions that favor the production of eriodictyol compositions by mixing two or more chemical or biological entities (such as compounds and enzymes). Means the process of interacting them below.

The term "degenerate variant" refers to a nucleic acid sequence that has a residue sequence that differs from the reference nucleic acid sequence due to one or more degenerate codon substitutions. Degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. The nucleic acid sequence and all degenerate variants thereof express the same amino acid or polypeptide.

The terms "polypeptide", "protein", and "peptide" give those skilled in the art their usual and customary meanings, and these three terms shall be used interchangeably. Also used without limitation to refer to amino acid polymers or amino acid analogs, regardless of size or function. Although "protein" is often used for relatively large polypeptides and "peptide" is often used for small polypeptides, the use of these terms in the art overlaps and varies. As used herein, the term "polypeptide" refers to peptides, polypeptides, and proteins, unless otherwise stated. The terms "protein", "polypeptide", and "peptide" are used interchangeably herein when referring to polynucleotide products. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs described above.

The terms "polypeptide fragment" and "fragment", when used with respect to reference polypeptides, give those skilled in the art their usual and conventional meaning, in which some amino acid residues are reference polypeptides. Although deleted relative to itself, the remaining amino acid sequence is typically used without limitation to refer to a polypeptide that is identical to the corresponding position of the reference polypeptide. Such deletions can occur at the amino-terminus and / or carboxy-terminus of the reference polypeptide.

The term "functional fragment" of a polypeptide or protein is part of a full-length polypeptide or protein and has substantially the same biological activity as, or substantially the same as, the full-length polypeptide or protein. Refers to peptide fragments that perform the same function (eg, perform the same enzymatic reaction).

The terms "mutant polypeptide," "modified amino acid sequence," or "modified polypeptide" used interchangeably by one or more amino acids, eg, substitutions, deletions, and / of one or more amino acids. Alternatively, it refers to an amino acid sequence that differs from the reference polypeptide by addition. In one aspect, the variant is a "functional variant" that retains some or all of the capabilities of the reference polypeptide.

The term "functional variant" further includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from the reference peptide by one or more conservative amino acid substitutions and maintains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is the substitution of an amino acid residue by a functionally similar residue. Examples of conservative substitutions are the replacement of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine with another; between arginine and lysine, between glutamine and asparagine, threonine and serine. Replacing one charged or polar (hydrophilic) residue with another residue, such as between; replacing one basic residue, such as lysine or arginine, with another; or asparagine This includes substituting an acidic residue such as acid or glutamate with another residue; or substituting an aromatic residue such as phenylalanine, tyrosine, tryptophan with another residue. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variant" means that certain residues have been chemically derivatized as long as the resulting peptide retains some or all of the activity of the reference peptides described herein. It also includes peptides that are substituted with residues.

The term "variant" associated with a polypeptide of the art refers to the amino acid sequence of the reference polypeptide and at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81. %, At least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, It further comprises a functionally active polypeptide having an amino acid sequence that is at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical.

The term "homology" in all its grammatical forms and spelling variations refers to polynucleotides or polypeptides having a "common evolutionary origin", including polynucleotides or polypeptides derived from the superfamily, and to different species. Refers to the relationship between the homologous polynucleotide or protein from which it is derived (Reeck et al., CELL 50: 667, 1987). Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, with respect to the percent identity or the presence of a particular amino acid or motif at a conserved position. For example, the two homologous polypeptides are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least. 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 900 at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least It can have amino acid sequences that are 98%, at least 99%, and even 100% identical.

The "appropriate regulatory sequence" gives those skilled in the art its usual and conventional meaning, upstream (5'non-coding sequence), internal, or downstream (3'non-coding sequence of coding sequence) of the coding sequence. It is used without limitation to refer to a nucleotide sequence that is located in and affects transcription, RNA processing or stability, or translation of the relevant coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"Promoter" is used to give one of ordinary skill in the art its usual and conventional meaning and is used without limitation to refer to a coding sequence or a DNA sequence capable of controlling the expression of functional RNA. Generally, the coding sequence is located at 3'of the promoter sequence. Promoters are either entirely derived from natural genes, or are composed of different elements from different promoters found in nature, or even contain synthetic DNA segments. Those skilled in the art will appreciate that different promoters can lead to gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental conditions. Promoters that, in most cases, cause gene expression in most cell types are commonly referred to as "constitutive promoters." Moreover, it is recognized that in most cases, DNA fragments of different lengths may have the same promoter activity, as the exact boundaries of the regulatory sequences are not completely defined.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that one function is influenced by the other. For example, a promoter is operably linked to a coding sequence if it can affect the expression of that coding sequence (ie, if the coding sequence is under transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in a sense or antisense orientation.

As used herein, the term "expression" gives those skilled in the art its usual and conventional meaning and is the transcription and stabilization of sense (mRNA) or antisense RNA derived from nucleic acid fragments of the art. Used without limitation to refer to accumulation. "Overexpression" refers to the production of a gene product in a transgenic or recombinant organism that exceeds the level of production in a normal or non-transformed organism.

"Transformation" gives one of ordinary skill in the art its usual and customary implications and is used without limitation to refer to the transfer of polynucleotides into target cells. The transferred polynucleotide can be integrated into the genome or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or it can replicate independently of the host chromosome. A host organism containing a transformed nucleic acid fragment may be referred to as "transgenic".

The terms "transformed," "transgenic," and "recombinant," as used herein in the context of a host cell, give those skilled in the art their usual and conventional meanings. There is and is used without limitation to refer to cells of a host organism such as plant or microbial cells into which a heterologous nucleic acid molecule has been introduced. Nucleic acid molecules can be stably integrated into the genome of the host cell, or nucleic acid molecules can exist as extrachromosomal molecules. Such extrachromosomal molecules can self-replicate. It is understood that a transformed cell, tissue, or subject includes not only the end product of the transformation process, but also its transgenic offspring.

The terms "recombinant," "heterologous," and "exogenous," as used herein in the context of polynucleotides, give those skilled in the art their usual and conventional meaning. Used without limitation to refer to a polynucleotide (eg, a DNA sequence or gene) from a foreign origin for a particular host cell, or a polynucleotide that has been modified from its original form if it comes from the same origin. .. Thus, heterologous genes in host cells include genes that are endogenous to a particular host cell but have been modified, for example, by site-specific mutagenesis or the use of other recombinant techniques. The term also includes multiple non-naturally occurring copies of naturally occurring DNA sequences. Thus, the term refers to a DNA segment that is exogenous or heterologous to a cell, or homologous to a cell, but in a location or morphology within the host cell where elements are not normally found.

Similarly, the terms "recombinant," "heterologous," and "exogenous," as used herein in connection with a polypeptide or amino acid sequence, are derived from a foreign origin for a particular host cell. It means a polypeptide or amino acid sequence to be used, or, if derived from the same origin, a polypeptide or amino acid sequence modified from its original form. Thus, recombinant DNA segments can be expressed in host cells to produce recombinant polypeptides.

The terms "plasmid," "vector," and "cassette" give those skilled in the art their usual and customary meanings and are not part of the central metabolism of the cell, but are usually circular double-stranded. It is used without limitation to refer to extra chromosomal elements that often carry genes that are in the form of DNA molecules. Such elements are of autonomously replicating sequences, genomic integration sequences, phage or nucleotide sequences, linear or circular sequences of single- or double-stranded DNA or RNA from any origin. Many nucleotide sequences, along with suitable 3'untranslated sequences, have been linked or recombined into unique structures that allow the introduction of promoter fragments and DNA sequences for selected gene products into cells. "Transformation cassette" refers to a specific vector containing a foreign gene and having an element that promotes transformation of a specific host cell in addition to the foreign gene. "Expression cassette" refers to a specific vector containing a foreign gene and having an element in addition to the foreign gene that allows the expression of the gene to be enhanced in the foreign host.

Synthetic Biology Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, in Sambrook, J. Mol. , Fritsch, E.I. F. And Maniatis, T. et al. MOLECULAR Cloning: A LABORATORY MANUAL, 2nd ed. Cold Spring Harbor Laboratory: Cold Spring Harbor, N.C. Y. , 1989 (hereinafter referred to as "Maniatis"); and Silhavey, T.I. J. , Bennan, M. et al. L. And Enquist, L .; W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.K. Y. , 1984; and Ausubel, F. et al. M. Et al., IN CURRETT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-InterScience, 1987; (the entire of which is incorporated herein by reference).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the practice or testing of the present disclosure, any method and material similar to or equivalent to that described herein can be used, but preferred materials and methods are described below.

This disclosure will be more fully understood in light of the following non-limiting examples. Although these examples show preferred embodiments of the subject art, it should be understood that they are given for illustration purposes only. From the above discussions and these examples, those skilled in the art will be able to identify the essential features of the subject technology and make various changes and modifications to the subject technology without departing from its spirit and scope. Can be adapted to various applications and conditions.

Materials and Methods Bacterial strains, plasmids, and culture conditions.
For DNA cloning and recombinant protein expression, E. coli of DH5a and BL21 (DE3). The E. coli strain was purchased from Invitrogen and the plasmids pRSFDuet-1 and pCDFDuet-1 were purchased from Novagen.

DNA manipulation.
All DNA manipulations were performed according to standard procedures. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. All PCR reactions were performed using the New England Biolabs Phaseion PCR system according to the manufacturer's guidance.

Identification of Target Genes SAM5, a 4-caumaric acid 3-hydroxylase derived from Saccharoslix espanaensis with NCBI reference sequence ID, WP_0151032334.1, first metahydroxylates from p-coumaric acid to caffeic acid. Catalyzing the formation was functionally characterized (Berner et al., 2006). The corresponding nucleotides are E. coli. After codon optimization for expression in E. coli, it was synthesized in the GenScript Company (SEQ ID NO: 1). To identify potentially interesting flavin reductase genes, the keyword "flavin reductase" was used to search the NCBI database for Pseudomonas fluorescent Pf-5 and Saccharos lix espanaensis proteins, respectively, AAY92875. Two homologues PfFR and SeFR with 1 GenBank ID and NCBI reference sequence of WP_041331262 were obtained. Their corresponding nucleotide sequences are described in E. coli. It was generated using codons optimized for expression in E. coli and synthesized with Genscript (NJ). E. HpaC (Galan et al. 2000), which encodes flavin reductase, which is a component of the 4-hydroxyphenylacetic acid hydroxylase complex of E. coli, was also included in this study.

Construction of plasmid.
DNA fragments of Sam5 and two uncharacterized flavin reductases, PpFR and SeFR, were presented in E. coli. Codon-optimized for expression in E. coli, these are associated with the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, respectively. The DNA sequence of natural HpaC is set forth in SEQ ID NO: 7. The corresponding protein sequences are set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, respectively. Sam5, PpFR, and SeFR were synthesized in the Genscript Company and used as templates for subsequent PCR amplification.

Sam5 was cloned into the NdeI / XhoI restriction site of pRSFDuet-1. PpFR, SeFR, and HpaC were cloned into the NdeI / XhoI restriction site of pCDFDuet-1. For overexpression and purification of the enzyme, HpaC, PpFR, and SeFR were cloned into the NdeI / XhoI restriction site of the pET28a vector.

HpaC is E.I. It was cloned from the genomic DNA of E. coli strain MG1655 and extracted using a bacterial DNA extraction kit. For the HpaC gene, the NdeI site was introduced at the 5'end and the XhoI site was introduced at the 3'end, and PCR was used to introduce E.I. It was amplified from E. coli genomic DNA. The primers used were the forward primer HpaC_NdeI_F (5'-GGGAATTCCATATGCAATTGAACAACGCCTGCG) and the reverse primer HpaC_XhoI_R (5'-CTCGAGCGGTTAAAATCGCAGCTTCCATTTCAGC). PCR products digested with Nde I and Xho I were ligated with the plasmid pCDFDuet-1 digested with the same enzyme, and E. coli was used. It was transformed into DH5a of E. coli. The plasmid HpaC-pCDF was confirmed by sequencing by extracting positive insertions from the colonies.

To create a SAM5 operon and a construct with a specific flavin reductase, a specific flavin reductase gene was inserted downstream of SAM5 by a Gibson assembly and SAM5-HpaC-pRSF, SAM5-PpFR-pRSF, and SAM5-SeFR- We obtained three constructs named pRSF.

E. using the developed structure. Transformation of E. coli BL21 (DE3).
Sam5-pRSF uses standard chemical transformation protocols to E. coli. Eriodictyol-producing E. coli introduced into E. coli BL21 (DE3) cells and named ERI-01. It led to the development of E. coli strain. Sam5-pRSF was co-transformed into BL21 (DE3) with PCDFDuet-1, PpFR-pCDF, SeFR-pCDF, and HpaC-pCDF, respectively, according to standard procedures, and the corresponding eriodictyol-producing E. coli. E. coli strains ERI-02, ERI-03, ERI-03, and ERI-05 were generated. Three plasmids with the constructed operons, SAM5-HpaC-pRSF, SAM5-PpFR-pRSF, and SAM5-SeFR-pRSF, were transformed into BL21 (DE3), respectively, and three E. coli. E. coli strains ERI-06, ERI-07, and ERI-08 were obtained.

E. Overexpression and purification of HpaC, PpFR, and SeFR in E. coli The plasmids, HpaC-pET28a, PpFR-pET28a, and SeFR-pET28a, respectively, are BL21 (DE3) competent for heterologous protein expression using standard procedures. Transformed into cells. Single colonies for each transformation were grown in 5 mL of LB medium with 50 mg / L kanamycin at 37 ° C. until OD600 reached about 1.0 and these seed cultures were accompanied by 50 mg / L kanamycin. Transferred to 200 mL of LB medium. The cells were grown at 37 ° C. and 250 rpm until the OD600 reached 0.6-0.8, then IPTG was added to a final concentration of 0.5 mM and the growth temperature was changed to 16 ° C. E. E. coli cells were harvested 16 hours after IPTG induction for protein purification by centrifugation at 4000 g for 15 minutes at 4 ° C. The resulting pellet was resuspended in 5 mL of 100 mM Tris-HCl, pH 7.4, 100 mM NaOH, 10% glycerol (v / v) and sonicated on ice for 2 minutes. The mixture was centrifuged at 4000 g for 20 minutes at 4 ° C. Recombinant proteins in the supernatant are prepared according to the manufacturer's protocol from Clontech Inc. Purified using His60 Ni Superflow resin.

Measurement of Flavin Reductase Activity Flavin reductase activity was determined by measuring the change in absorbance at 340 nm at 30 ° C. using SpectraMax i3. A fixed amount of purified protein (0.1 μg each) was incubated with 400 μM NADH and 200 μM FAD in reaction buffer (Tris-HCl 20 mM pH 7.4, final volume 100 μL). The assay mixture without FAD was used as a blank.

Biological Conversion of Naringenin to Eriodictyol The present invention allows the modified microbial strains of the present invention to catalyze the conversion of naringenin to eriodictyol with high efficiency, along with SAM5. We have developed a system that overexpresses flavin reductase. The titer of the shaking flask reached 0.65 g / L in 6 hours. The microbial system used was E. coli. E. coli BL21 (DE3) strains, ERI-01, ERI-06, ERI-07, ERI-08, and pRSF-BLK, were grown on LB medium containing 30 μg / L kanamycin. ERI-02, ERI-03, ERI-03, and ERI-05 were grown on LB medium containing 30 μg / L kanamycin and 100 μg / L spectinomycin, respectively. After growing the cells in a shaker at 37 ° C. to OD600 = 0.6, lactose was added to a final concentration of 1.5% (w / v) to change the temperature to 30 ° C. to induce expression of exogenous genes. .. Three hours after induction of expression, naringenin (40% w / v) dissolved in DMSO was added to the culture. The culture continued to shake under the same culture conditions. Samples were taken 6 hours after feeding the substrate for HPLC analysis.

HPLC and LC-MS analysis.
HPLC analysis of flavonoids was performed on the Dionex Ultimate 3000 system. The intermediate uses a gradient of 0.15% (vol / vol) acetic acid (eluent A) and acetonitrile (eluent B), with eluent B in the range of 10-40% (vol / vol). Separation was performed by reverse phase chromatography on a Dionex Acclim 120 C18 column (particle size 3 mm; 150 x 2.1 mm) at a flow rate of 0.6 ml / min. For quantification, all intermediates were calibrated using external standards. The compounds were identified by their retention time and the corresponding spectrum identified by the diode array detector in the system.

For DNA cloning and recombinant protein expression, E. coli of DH5a and BL21 (DE3). The E. coli strain was purchased from Invitrogen and the plasmids pRSFDuet-1 and pCDFDuet-1 were purchased from Novagen.

Production System In one embodiment, the expression vector comprises those genetic elements for the expression of recombinant polypeptides in bacterial cells. Elements for transcription and translation in bacterial cells include promoters, coding regions of protein complexes, and transcription terminators.

Those of skill in the art are aware of the molecular biology techniques available for the preparation of expression vectors. As mentioned above, the polynucleotides used to incorporate the subject technology into expression vectors can be prepared by routine techniques such as the polymerase chain reaction (PCR). In molecular cloning, a vector is a DNA molecule used as a medium for artificially transporting a foreign genetic material to another cell, where the vector can replicate and / or express (plasmid, cosmid, lambda phage). Such). Vectors containing foreign DNA are considered recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vector is a plasmid. Common to all engineered vectors are the origin of replication, the multicloning site, and selectable markers.

Many molecular biology techniques have been developed to operably link DNA to vectors via complementary attachment ends. In one embodiment, a bundle of complementary homopolymers can be added to the nucleic acid molecule and inserted into the vector DNA. The vector and nucleic acid molecules are then bound by hydrogen bonds between the complementary homopolymer tails to form recombinant DNA molecules.

In another embodiment, a synthetic linker containing one or more restriction site grants is used to operably link the polynucleotide of the subject art to an expression vector. In one embodiment, the polynucleotide is produced by restriction endonuclease digestion. In one embodiment, the nucleic acid molecule uses the enzyme's own 3'-5'exonuclease activity to remove the protruding 3'single strand end and the enzyme's own polymerase activity to remove the recessed 3'end. Bacterophage T4 DNA polymerase or E. coli, an enzyme that fills the It is processed with E. coli DNA polymerase I, which produces blunt-ended DNA segments. The blunt-ended segment is then incubated with a molar excess of linker molecules in the presence of an enzyme capable of catalyzing the ligation of the blunt-ended DNA molecule, such as bacteriophage T4 DNA ligase. Therefore, the product of the reaction is a polynucleotide having a polymer linker sequence at its end. These polynucleotides are then cleaved with a suitable restriction enzyme and ligated into an expression vector cleaved with an enzyme that produces an end that matches the end of the polynucleotide.

Alternatively, a vector having a ligation-independent cloning (LIC) site can be used. The required PCR-amplified polynucleotide can then be cloned into a LIC vector without restriction digestion or ligation (Aslanidis and de Jong, NUCL.ACID.RES18 6069-74, (1990), Haun et al., BIOTECHNIQUES 13 , 515-18 (1992), each of which is incorporated herein by reference).

In one embodiment, it is appropriate to use PCR to isolate and / or modify the polynucleotide of interest for insertion into the selected plasmid. Design suitable primers for use in PCR preparation of sequences, isolate the required coding region of the nucleic acid molecule, add restriction endonucleases or LIC sites, and place the coding region within the desired reading frame. can do.

In one embodiment, the polynucleotide for incorporation into the expression vector of the technique is prepared using oligonucleotide primers suitable for PCR. The coding region is amplified, but the primers themselves are incorporated into the amplified sequence product. In one embodiment, the amplification primer contains a restriction endonuclease recognition site, which allows the amplified sequence product to be cloned into a suitable vector.

Expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Appropriate cell transformation with expression vectors of the subject technology is accomplished by methods known in the art and typically depends on both vector and cell types. Suitable techniques include coprecipitation of calcium phosphate or calcium chloride, DEAE-dextran-mediated transfection, lipofection, chemical perforation, or electroporation.

Successfully transformed cells, i.e. cells containing an expression vector, can be identified by techniques well known in the art. For example, cells transfected with the expression vector of the present technology can be cultured to produce the polypeptides described herein. Cells can be tested for the presence of expression vector DNA by techniques well known in the art.

The host cell can include a single copy of the expression vector described above, or, optionally, multiple copies of the expression vector.

In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algae cell, a fungal cell, or a yeast cell. In some embodiments, the cells are canola plant cells, rapeseed plant cells, palm plant cells, sunflower plant cells, cotton plant cells, corn plant cells, peanut plant cells, flax plant cells, sesame plant cells, soybean plant cells, It is a plant cell selected from the group consisting of petunia plant cells.

Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high levels of expression of foreign proteins well known to those of skill in the art. Any of these could be used to construct a vector for the expression of recombinant polypeptides of the subject technology in microbial host cells. These vectors could then be introduced into the appropriate microorganism by transformation to allow high levels of expression of the recombinant polypeptides of the subject technology.

Vectors or cassettes useful for transformation of suitable microbial host cells are well known in the art. Usually, the vector or cassette contains sequences that direct transcription and translation of the relevant polynucleotide, selectable markers, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors include a 5'region of a polynucleotide that controls transcription initiation and a 3'region of a DNA fragment that controls transcription termination. Both regulatory regions are preferably derived from genes homologous to the transformed host cell, but such regulatory regions need not be derived from genes specific to the particular species selected as the host. I want you to understand.

The termination control region may be derived from various genes specific to the microbial host. Termination sites can optionally be included for the microbial hosts described herein.

Biosynthesis and Use of Flavonoids of the Invention Those skilled in the art will further purify the eriodictyol compositions produced by the methods described herein to further purify other dietary supplements, pharmaceutical compositions, functional cosmetics, nutrients. , As well as can be mixed with pharmaceutical products.

Analysis of Sequence Similarity Using Identity Scoring As used herein, "sequence identity" is the alignment of two optimally aligned polynucleotide or peptide sequences to their constituents, such as nucleotides or amino acids. It is immutable and points to some extent across windows. The "identity ratio" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences, that is, the total number of components in the reference sequence segment. , All of the reference sequence or divided by the smaller demarcated portion of the reference sequence.

As used herein, the term "percent sequence identity" or "percent identity" is the appropriate nucleotides in the total number of less than 20% of the reference sequence when the two sequences are optimally aligned (over the comparison window). A linear polynucleotide of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand), with insertions, deletions or gaps. Refers to the proportion of identical nucleotides in a sequence. Optimal sequence alignment for aligning comparison windows is well known to those skilled in the art and includes local homology algorithms for Smith and Waterman, homology alignment algorithms for Needleman and Wunsch, methods for searching for Pearson and Lipman similarities, and the like. These algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, which can be implemented by tools and are preferably available as part of GCG® WisconsinPackage® (Accelrys Inc., Burlington, MA). It can be implemented by a computerized implementation of. The "identity ratio" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences, that is, the total number of components in the reference sequence segment. , All of the reference sequence or divided by the smaller demarcated portion of the reference sequence. The percent sequence identity is expressed as the percentage of identity multiplied by 100. Comparison of one or more polynucleotide sequences can be for full-length polynucleotide sequences or parts thereof, or longer polynucleotide sequences. For the purposes of the present invention, the "percent identity" can also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

The percentage of sequence identity is preferably determined using the "Best Fit" or "Gap" program of the Sequence Analysis Software Package ™ (version 10; Genetics Computer Group, Inc., Madison, WI). "Gap" uses Needleman and Wunch algorithms (Needleman and Wunch, JOURNAL OF MOLECULAR BIOLOGY 48: 443-453, 1970) to align two sequences that maximize the number of matches and minimize the number of gaps. locate. "BestFit" uses Smith and Waterman's local homology algorithms (Smith and Waterman, ADVANCES IN APPLIED MATHEMATICS, 2: 482-489, 1981, Smith et al., nucleoIC ACIDS RESEARCH 11: 2205-2220, 198). Optimal alignment of the best segment is performed for the similarity between the two sequences and the insertion gap to maximize the number of matches. The percent identity is most preferably determined using the "Best Fit" program.

Useful methods for determining sequence identity are described in National Library of Medicine, National Institutes of Health, Bethesda, Md. It is disclosed in the Basic Local Element Search Tool (BLAST), which is publicly available from the National Center for Biotechnology Information (NCBI) at 20894; BLAST Manual, Altschul et al., NCBI, N. MOL. BIOL. 215: 403-410 (1990); since version 2.0 of the BLAST program, gaps (deletion and insertion) can be introduced into the alignment; for peptide sequences, sequence identity using BLASTX. And for polynucleotide sequences, BLASTN can be used to determine sequence identity.

As used herein, the term "substantial percentage of sequence identity" refers to at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, and at least about 90. % Of sequence identity, or even greater sequence identity, eg, about 98% or about 99% of sequence identity. Thus, one embodiment of the invention is at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% identity, at least about 90% with the polynucleotide sequences described herein. A polynucleotide molecule having a sequence identity of, or even greater sequence identity, eg, about 98% or about 99% sequence identity. The polynucleotide molecule carrying the active gene of the invention can direct the production of eriodicthiol and has a substantial percent sequence identity to the polynucleotide sequence provided herein, the invention. Is included in the range of.

Identity and Similarity Identity is the percentage of amino acids that are the same between a pair of sequences after sequence alignment (this is done using only sequence or structural information or some other information). (Although it can be done, it is usually based solely on sequence information), similarity is a score assigned based on alignment using several similarity matrices. The similarity indicator can be any one of the following BLOSUM62, PAM250, or GONNET, or any matrix used by one of ordinary skill in the art for protein sequence alignment.

Identity is the degree of matching between two subsequences (no gaps between sequences). More than 25% of identities imply functional similarities and 18-25% imply structural or functional similarities. It should also be noted that two completely unrelated or random sequences (more than 100 residues) can have more than 20% identity. Similarity is the degree of similarity between two sequences when compared. This depends on their identity.

As will be apparent from the above description, certain aspects of the present disclosure will not be limited by the particular detailed description of the examples provided herein, and thus other modifications and applications, or equivalents thereof. Is assumed to those skilled in the art. Therefore, the scope of claims shall cover all such amendments and applications that do not deviate from the spirit and scope of the present disclosure.

Moreover, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the practice or testing of the present disclosure, any method and material similar to or equivalent to that described herein can be used, but preferred methods and materials are described above.

Although the invention described above has been described in some detail by way of example and examples for the purpose of understanding, it will be apparent to those skilled in the art that certain modifications and modifications may be made. Therefore, this description and examples should not be construed as limiting the scope of the invention as defined by the appended claims.

Results Sam5 activity in the biological conversion of naringenin to eriodictyol As shown in FIG. 1, Sam5 was inserted into the second polycloning site of pRSFDuet-1. Gene expression is under the control of the T7 promoter. When the plasmid Sam5-pRSF was introduced into BL21 (DE3), the cells had the ability to convert naringenin fed to the culture to eriodictyol, but a control carrying the Sam5-free pRSFDuet-1 plasmid. No eriodictyol was produced in the strain ERI-Ctrl. After feeding the cell culture with naringenin, a small amount of new compound was produced, as shown in the HPLC profile. The retention time and absorption spectrum of this compound in HPLC analysis corresponds to eriodictyol. Further analysis of the peaks separated by HPLC as shown in FIG. 3 confirmed the biological conversion of naringenin to eriodictyol.

PpFR and SeFR are flavin reductases.
HpaC is E.I. It encodes flavin reductase, a component of the 4-hydroxyphenylacetic acid hydroxylase complex of E. coli (Galan et al., 2000). The other two genes, PpFR and SeFR, were identified from the NCBI database in this study. In sequence analysis, both share low identity with HpaC, 22.9% and 30.2%, respectively. However, each of these three proteins carries the S / T / CxxPP and DGH consensus motifs characteristic of the HpaC-like subfamily of class I flavin reductase (Fig. 4). Therefore, PpFR and seFR were annotated as members of the HpaC-like subfamily of class I flavin reductase (Van Lanen et al., 2009). All three of these genes were cloned into the pET28a vector (Fig. 7), respectively, due to overexpression. It was introduced in E. coli BL21 (DE3). Recombinant protein E. It was expressed in E. coli and purified to homogeneity for biochemical analysis (Fig. 5). The results clearly confirmed the above bioinformatics analysis. Similar to HpaC, PpFR and SeFR catalyze the reduction of FAD by NADH, and the specific activity of PpFR and SeFR is comparable to that of HpaC (Fig. 6).

Flavin reductase dramatically increases the bioconversion of naringenin to eriodictyol by Sam5.
HpaC-pCDF was converted to E.I. When co-expressed with Sam5-pRSF in E. coli cells, the resulting engineered cell ERI-03 can convert naringenin to eriodictyol with much higher activity in vivo. As shown in FIG. 8, the eriodictyol produced reached much higher levels than that produced by SAM5 alone. Eriodictyol production with the ER03 strain reached 0.285 g / L in 6 hours, which was 6.3 times and 7.5 times that of production with the ERI-01 and ERI-02 strains, respectively. This is a double increase (Fig. 9). PpFR and SeFR also showed a stimulating effect on SAM5. ERI-04 and ERI-05 have been shown to have high activity to catalyze the hydroxylation of naringenin to produce eriodictyol. As shown in FIG. 9, 6 hours after feeding naringenin to ERI-04 and ERI-05, respectively, eriodictyol accumulated 357 mg / L and 388 mg / L in the cell culture medium, respectively. Compared to that of ERI03, the titers of eriodictyol produced by ERI-04 and ERI-05 were increased by 25% and 36%, respectively (Fig. 9). This result suggests that PpFR and SeFR may be more effective in promoting sam5-catalyzed hydroxylation of naringenin.

The SAM5 and flavin reductase operons further increased the biological conversion of naringenin to eriodictyol.
As shown in FIG. 8, the eriodictyol produced with the ERI-02 strain is lower than that with the ERI-01 strain, which can be caused by the two antibodies used in the co-expressing strain. There is sex. SAM5 and flavin reductase were constructed for the expression vector operon (Fig. 9). The obtained E. The E. coli strains ERI-06, ERI07, and ERI-08 were tested for the bioconversion of naringenin to eriodictyol. As shown in FIG. 11, the titers reached 589, 614, and 638 mg / L, which were significantly higher than the corresponding co-expressing strains.

Use of Functional Cosmetics and Dietary Supplements Molecular biology plays a vital role in the innovation of functional cosmetics. Here, the identification of a compound begins with the identification of a molecular target. For example, the importance of free radicals associated with skin aging has in recent years led to a thorough search for active substances that eliminate the harmful effects of free radicals and thereby protect tissues from oxidative damage. Skin aging manifests as age spots, more specifically melasma, dyschromia, melanoma, and wrinkles, predominantly free for tissues that cause cross-linking and glycation of structural proteins, as well as pro-inflammatory enzyme systems Due to radical damage. The use of flavonoids in cosmetics or pharmacies is known in itself. Natural antioxidants that suppress free radicals, such as eriodictyol of the present invention, are essential components of anti-aging formulations. They potentially provide protection against tissue damage and the harmful effects of the environment and other drugs. Biochemical reactions that accelerate the progression of skin aging have a root cause in the inflammatory process, because inflammation produces microscarring that develops into scars or wrinkles.

These flavones and flavonic glycoside derivatives (flavonoids) discussed herein are known to be inhibitors of oxygen radical scavengers and skin proteases that can actively combat skin aging and scarring. ing. Some flavones, such as quercetin, are also useful as food colorants due to their coloring properties. At the same time, it can also be used as an antioxidant due to its ability to capture oxygen radicals. Some flavonoids are inhibitors of aldose reductase that play an important role in the formation of diabetic damage (eg, vascular damage). Other flavonoids (such as hesperidin and rutin) are used therapeutically, more specifically as vasodilators.

Scientific studies have confirmed the widespread effects of flavonoid compounds on the skin at various levels. The stratum corneum, the top layer of the skin, is a structure very rich in lipids and other oxidizable compounds. In this layer, flavonoids can play an efficient role as antioxidants and free radical scavengers. Due to their antioxidant properties, they affect the deeper epidermal skin layer, prevent UV damage and inhibit some enzymatic functions. In the deepest layer of the skin, the dermis, flavonoids affect the permeability and fragility of the microvascular system. The valuable features of flavonoids mentioned above are already of value to the cosmetics industry.

Industrial Applicability / Technical Field Statements This disclosure is applicable to the food, feed, functional cosmetics, and pharmacological industries. The present disclosure generally relates to methods for the biosynthetic production of eriodictyol via a modified microbial strain.
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25.
Target sequence SEQ ID NO 1: SAM5 of the nucleic acid sequence ATGACGATTACCTCTCCGGCCCCGGCTGGTCGCCTGAACAATGTGCGTCCGATGACGGGTGAAGAATACCTGGAATCCCTGCGTGACGGTCGTGAAGTGTATATTTACGGCGAACGCGTCGATGACGTGACCACGCATCTGGCGTTCCGCAACAGCGTTCGTTCTATCGCCCGCCTGTATGATGTCCTGCACGATCCGGCCTCCGAAGGTGTTCTGCGCGTCCCGACCGATACCGGTAATGGTGGTTTTACCCATCCGTTTTTCAAAACGGCGCGTAGCTCTGAAGACCTGGTGGCGGCCCGTGAAGCCATTGTCGGTTGGCAACGCCTGGTGTATGGCTGGATGGGTCGTACCCCGGATTACAAGGCAGCGTTTTTCGGTACGCTGGACGCTAACGCGGAATTTTATGGCCCGTTCGAAGCCAATGCACGTCGCTGGTATCGTGATGCACAGGAACGCGTTCTGTACTTCAACCATGCTATCGTGCATCCGCCGGTCGATCGTGACCGTCCGGCTGATCGTACCGCCGACATTTGCGTCCATGTGGAAGAAGAAACGGATTCAGGCCTGATCGTGTCGGGTGCCAAAGTGGTTGCAACCGGTTCTGCTATGACGAACGCGAATCTGATTGCCCACTATGGTCTGCCGGTTCGCGATAAAAAGTTTGGCCTGGTGTTCACCGTTCCGATGAACAGTCCGGGTCTGAAACTGATCTGTCGTACCTCCTATGAACTGATGGTGGCCACGCAGGGCTCACCGTTTGATTACCCGCTGAGTTCCCGCCTGGATGAAAATGACAGCATTATGATCTTTGATCGTGTTCTGGTCCCGTGGGAAAACGTTTTCATGTACGACGCAGGCGCGGCCAATAGCTTTGCTACCGGCTCTGGTTTCCTGGAACGCTTTACCTTTCATGGCTGCACGCGTCTGGCAGTGAAACTGGATTTTATTGCAGGCTGTGTTATGAAGGCTGTGGAAGT TACCGGCACCACGCACTTCCGCGGTGTTCAGGCGCAAGTCGGCGAAGTGCTGAACTGGCGTGATGTCTTTTGGGGTCTGTCGGACGCTATGGCGAAAAGTCCGAACAGCTGGGTGGGCGGTAGCGTTCAGCCGAACCTGAATTATGGCCTGGCCTACCGCACCTTTATGGGCGTGGGTTATCCGCGTATTAAAGAAATTATCCAGCAAACGCTGGGCTCTGGTCTGATCTACCTGAACTCATCGGCAGCTGATTGGAAGAATCCGGACGTTCGCCCGTATCTGGATCGTTACCTGCGCGGCAGTCGTGGTATTCAGGCAATCGATCGTGTCAAACTGCTGAAGCTGCTGTGGGACGCGGTGGGCACCGAATTTGCCGGTCGTCATGAACTGTATGAACGCAACTACGGCGGTGATCACGAAGGCATTCGTGTGCAGACCCTGCAAGCCTATCAGGCAAATGGTCAAGCGGCGGCACTGAAAGGCTTTGCGGAACAGTGCATGAGCGAATACGACCTGGATGGCTGGACCCGCCCGGACCTGATTAACCCGGGCACCTGA
The amino acid sequence MTITSPAPAGRLNNVRPMTGEEYLESLRDGREVYIYGERVDDVTTHLAFRNSVRSIARLYDVLHDPASEGVLRVPTDTGNGGFTHPFFKTARSSEDLVAAREAIVGWQRLVYGWMGRTPDYKAAFFGTLDANAEFYGPFEANARRWYRDAQERVLYFNHAIVHPPVDRDRPADRTADICVHVEEETDSGLIVSGAKVVATGSAMTNANLIAHYGLPVRDKKFGLVFTVPMNSPGLKLICRTSYELMVATQGSPFDYPLSSRLDENDSIMIFDRVLVPWENVFMYDAGAANSFATGSGFLERFTFHGCTRLAVKLDFIAGCVMKAVEVTGTTHFRGVQAQVGEVLNWRDVFWGLSDAMAKSPNSWVGGSVQPNLNYGLAYRTFMGVGYPRIKEIIQQTLGSGLIYLNSSAADWKNPDVRPYLDRYLRGSRGIQAIDRVKLLKLLWDAVGTEFAGRHELYERNYGGDHEGIRVQTLQAYQANGQAAALKGFAEQCMSEYDLDGWTRPDLINPGT of SEQ ID NO: 2 SAM5.
Nucleic acid sequence ATGATGACCGTTTATGATAGCGCACTGACAATGGAAGAAACCACCCTGCGTGATGCAATGAGCCGTTTTGCAACCGGTGTTAGCGTTGTTACCGTTGGTGGTGAACATACACATGGTATGACCGCAAATGCCTTTACCTGTGTTAGCCTGGATCCGCCTCTGGTTCTGTGTTGTGTTGCACGTAAAGCAACCATGCATGCAGCAATTGAAGGTGCACGTCGTTTTGCAGTTAGCGTTATGGGTGGTGATCAAGAACGTACCGCACGTTATTTTGCAGATAAACGTCGTCCGCGTGGTCGTGCACAGTTTGATGTTGTTGATTGGCAGCCTGGTCCGCATACAGGTGCACCGCTGCTGAGCGGTGCGCTGGCATGGCTGGAATGTGAAGTTGCACAGTGGCATGAAGGTGGCGATCATACCATTTTTCTGGGTCGTGTTCTGGGTTGTCGTCGTGGTCCGGATAGTCCGGCACTGCTGTTTTATGGTAGCGATTTTCATCAGATCCGCTAA of SEQ ID NO: 3 SeFR
Amino acid sequence of SEQ ID NO: 4 SeFR MMTVYDSALTMETTLRRDAMSRFATGVSVVTVGGGEHTHGMTANAFTCVSLDPPLVLCCVARKATMHAAIEGARRFAVSVMGGDQERTARYFADKRRPRGRGRAGRAQSFGLVVGRFLGFLGFGFRGG
SEQ ID NO: 5 5 PfFR of the nucleic acid sequence ATGAATGCAGCAACCGAAACCAAAGTTCATGATCTGCTGGATGCCGAAGGTCGTGATGTTCGTGATGCACGTGAACTGCGTAATGTTCTGGGTCAGTTTGCAACCGGTGTTACCGTTATTACCACCCGTACCGCAGATGGTCGTAATGTTGGTGTGACCGCAAATAGCTTTAGCAGCCTGAGCCTGAGTCCGGCACTGGTTCTGTGGTCACTGGCACGTACCGCACCGAGCCTGAAAGTTTTTTGTAGCGCAAGCCATTTTGCCATTAATGTGCTGGGTGCACATCAGCTGCATCTGAGCGAACAGTTTGCACGTGCCGCAGCAGATAAATTTGCCGGTGTTGCACATAGTTATGGTAAAGCGGGTGCACCGGTTCTGGATGATGTTGTTGCAGTTCTGGTTTGCCGTAATGTTACCCAGTATGAAGGTGGTGATCATCTGATTTTTATCGGCGAAATTGAGCAGTATCGTTATAGCGGTGCAGAACCGCTGGTTTTTCATGCAGGTCAGTATCGTGGTCTGGGTAGCAATCGTGCAGAAAGCGTTCTGAAACATGAATAA
The amino acid sequence MNAATETKVHDLLDAEGRDVRDARELRNVLGQFATGVTVITTRTADGRNVGVTANSFSSLSLSPALVLWSLARTAPSLKVFCSASHFAINVLGAHQLHLSEQFARAAADKFAGVAHSYGKAGAPVLDDVVAVLVCRNVTQYEGGDHLIFIGEIEQYRYSGAEPLVFHAGQYRGLGSNRAESVLKHE of SEQ ID NO: 6 PfFR.
Nucleic acid sequence ATGCAATTAGATGAACAACGCCTGCGCTTTCGTGACGCAATGGCCAGCCTGTCGGCAGCGGTAAATATTATCACCACCGAGGGCGACGCCGGACAATGCGGGATTACGGCAACGGCCGTCTGCTCGGTCACGGATACACCACCATCGCTGATGGTGTGCATTAACGCCAACAGTGCGATGAACCCGGTTTTTCAGGGCAACGGTAAGTTGTGCGTCAACGTCCTCAACCATGAGCAGGAACTGATGGCACGCCACTTCGCGGGCATGACAGGCATGGCGATGGAAGAGCGTTTTAGCCTCTCATGCTGGCAAAAAGGTCCGCTGGCGCAGCCGGTGCTAAAAGGTTCGCTGGCCAGTCTTGAAGGTGAGATCCGCGATGTGCAGGCAATTGGCACACATCTGGTGTATCTGGTGGAGATTAAAAACATCATCCTCAGTGCAGAAGGTCACGGACTTATCTACTTTAAACGCCGTTTCCATCCGGTGATGCTGGAAATGGAAGCTGCGATTTAA of SEQ ID NO: 7 HpaC
Amino acid sequence of SEQ ID NO: 8 HpaC MQLDEQRLRFRRDAMASLSAAVNIITTEGDAGQCGITATAAVCSVTTDTPPSLMVCINANSAMNPVFQGNGKLCVNVLNHEQELMARHFAGMTGMEMERFSLSCWQKGLAQPLVLIVAL

Claims (48)

An isolated recombinant host cell transformed with a nucleic acid construct containing a polynucleotide sequence encoding a flavin reductase operably linked to one or more control sequences, wherein the flavin reductase is the hydroxyl of naringenin. An isolated recombinant host cell capable of directing biological conversion to flavonoids. The isolated recombinant cell according to claim 1, further comprising a vector containing the isolated nucleic acid sequence of SEQ ID NOs: 1, 3, 5, and 7. The isolated recombinant cell of claim 1, further comprising a vector containing the isolated nucleic acid sequence of SEQ ID NOs: 1 and 3. The isolated recombinant cell according to claim 1, further comprising a vector containing the isolated nucleic acid sequence of SEQ ID NOs: 1 and 5. The isolated recombinant cell according to claim 1, further comprising a vector containing the isolated nucleic acid sequence of SEQ ID NOs: 1 and 7. The isolated recombinant cell of claim 1, further comprising the isolated nucleic acid sequence of SEQ ID NO: 3. The isolated recombinant cell of claim 1, wherein the polynucleotide comprises a sequence that is at least 90% identical to the DNA sequence of SEQ ID NO: 3. The isolated recombinant cell of claim 1, further comprising the isolated nucleic acid sequence of SEQ ID NO: 5. The isolated recombinant cell of claim 1, wherein the polynucleotide comprises a sequence that is at least 90% identical to the DNA sequence of SEQ ID NO: 5. The isolated recombinant cell of claim 1, further comprising the isolated nucleic acid sequence of SEQ ID NO: 7. The isolated recombinant cell of claim 1, wherein the polynucleotide comprises a sequence that is at least 90% identical to the DNA sequence of SEQ ID NO: 7. The isolated recombinant cell of claim 1, wherein the flavin reductase comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence from SEQ ID NO: 4. The isolated recombinant cell of claim 1, wherein the flavin reductase comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence from SEQ ID NO: 6. The isolated recombinant cell of claim 1, wherein the flavin reductase comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence from SEQ ID NO: 8. The isolated recombinant cell according to claim 1, wherein the hydroxylated flavanoid is eriodictyol. The isolated recombinant cell according to claim 1, wherein the isolated recombinant cell is selected from the group consisting of bacteria, yeast, filamentous fungi, cyanobacteria, and plant cells. The isolated recombinant cells are Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Corynebacterium; Corynebacterium; Corynebacterium; (Methylomonas); Rhodococcus (Rhodococcus); Pseudomonas (Pseudomonas); Rhodobacter (Rhodobacter); Synechocystis (Synechocystis); Saccharomyces (Saccharomyces); Zygo Saccharomyces (Zygosaccharomyces); Kluyveromyces (Kluyveromyces); Candida (Candida); Hansenula (Hansenula); Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotris; Brevibacterium; Brevibacteria; Brevibacteria; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; from Clostridium; from Clostridium; from Clostridium; and from Clostridium; , The isolated recombinant cell according to claim 1. A method for producing an eriodictyol composition, which comprises incubating the isolated recombinant host cell according to claim 15 with a substrate. The eriodictyol composition produced by the method according to claim 18. The method of claim 18, wherein the substrate is a naringenin composition, wherein the naringenin is hydroxylated to enzymatically form eriodictyol. The isolated recombinant cell according to claim 1, wherein the isolated recombinant cell is selected from the group consisting of yeast, non-eriodictyol-producing plants, algae, and bacteria. The isolated recombinant cells are E. coli. The isolated recombinant cell according to claim 16, which is an E. coli cell. Low containing water, a non-nutritive sweetener component including a non-nutritive sweetener having a prolonged sweet aftertaste, and an amount of a bitter component effective in reducing the prolonged sweet aftertaste of the non-nutritive sweetener. A low-calorie beverage product, wherein the bitterness component is eriodicthiol produced by the method according to claim 18. The low-calorie beverage according to claim 23, wherein the concentration of the bitterness component is 50 ppt (1 trillion percent) to 500 ppm (parts per million). The low-calorie beverage according to claim 23, further comprising rebaugioside M. A method of using natural compounds in cosmetic or hygienic products, where a step of producing eriodicthio from microbial host cells and the combination of the eriodicthio with a cosmetically or hygienic active substance is desired. A method comprising the process of manufacturing a cosmetic or hygienic product by providing a skin, hair, or oral care effect. A cosmetic or hygienic composition produced by the method of claim 26. The cosmetic or hygienic composition according to claim 27, wherein the composition has strong anti-inflammatory activity. The cosmetic or hygienic composition according to claim 28, wherein the composition is utilized as an agent for enhancing bioavailability. The cosmetic or hygienic composition of claim 29, further comprising Vitamin C. 29. The cosmetic or cosmetic product according to claim 29, wherein the composition has a free radical degrading effect so that the composition reduces at least one of skin damage, hair damage, and oral damage caused by free radicals. Sanitary composition. 26. The method of claim 26, wherein the eriodictyol component is purified to a purity of at least 50%. 32. The method of claim 32, wherein the eriodictyol has a purity greater than 70%. 26. The method of claim 26, wherein the content of eriodictyol is greater than about 85% by weight based on dry mass. i) Purify the eriodictyol from the isolated recombinant cells to produce a crude product, and ii) remove the solvent under vacuum to provide a concentrated riodictyol product. The method of claim 18, further comprising: 35. The method of claim 35, wherein the crude product is purified by column chromatography. 35. The method of claim 35, wherein the crude product is purified by acid-base extraction. 35. The method of claim 35, wherein the crude product is purified by vacuum distillation. 35. The method of claim 35, further comprising purifying the eriodictyol using semi-preparative HPLC. The isolated recombinant host cell is E. coli. It is an E. coli cell, and the above method is E. coli. The method of claim 18, further comprising co-expressing HpaC with Sam5 in E. coli cells, wherein the resulting cells convert naringenin to eriodictyol. The isolated recombinant cell according to claim 1, wherein the isolated recombinant cell is a Pichia Pastoris cell. The isolated recombinant cell according to claim 1, wherein the isolated recombinant cell is a Saccharomyces Cerevisiae cell. The method according to claim 18, wherein the eriodictyol composition produced is eriodictyol, pinocembrin, homoeriodictyol, or a combination thereof. The eriodictyol compound is incorporated into a food as a sodium salt or potassium salt in the range of 0.01 to 500 ppm, and acts to reduce or suppress the bitterness in the impression of the food without reducing the sweetness. , The eriodictyol composition according to claim 19. The eriodictyol composition according to claim 44, wherein the sweetness of the food is provided by Stevia rebaugioside. The eriodictyol composition according to claim 44, wherein the eriodictyol composition is accompanied by another flavoring agent, and the flavoring agent is selected from the group containing trilobine, sakuranetin, phloretin, and hesperetin. The eriodictyol composition produced can suppress bitterness, and the bitterness suppressed by the eriodictyol composition is present in foods, dietary supplements, pharmaceuticals, consumables, pharmaceutical products, and oral hygiene. It is in a preparation selected from the group consisting of products for use, and in some cases, the method comprises adding the riodictyol composition to a food, dietary supplement, pharmaceutical, consumable beverage, pharmaceutical product, or oral hygiene product. The method of claim 18, further comprising adding. The isolated recombinant cells are soybean; rapeseed; sunflower; cotton; corn; tobacco; alfalfa; wheat; barley; oats; sorghum; rice; broccoli; potash flower; cabbage; parsnip; melon; carrot; celery; parsley. Tomatoes; potatoes; strawberries; peanuts; grapes; grass seed grains; tensai; sugar cane; legumes; pea beans; rye; flax; hardwood; softwood; feed grass; sorghum (Arabidopsis thaliana); rice From a group consisting of barley (Hordeum yulgare); switchgrass (Panicum vigratum); sorghum sp (Brachypodium spp); abrana (Brassica spp.); The isolated recombinant cell according to claim 1.